Virus assembly occurs in the complex three-dimensional environment of the cell, and new advances in electron microscopy tomography (EMT) now make it possible to construct high-resolution three-dimensional images where the spatial location of components involved in virus assembly can be determined. The simplicity of the Flock House virus (FHV) capsid and the extensive structural, biochemical, and genetic characterization of the virus make it an excellent system for studying in situ virus assembly. The focus of this proposal is to use to EMT to answer key questions about Flock House virus assembly in the cell and then extend these approaches to Influenza virus genome packaging and assembly. Aim 1: Three-dimensional imaging of the Flock House virus factories in vitro and in situ. Our hypothesis is FHV assembles in viral factories, which are formed from extensively modified mitochondria and are then trafficked on cytoskeleton to viral arrays. The modified mitochondria will be isolated and imaged using cryo-EMT to obtain near native state samples. The viral factories will then be imaged using cellular EMT to understand the virus assembly process in the context of the cell. Aim 2: EM-tomography analysis of influenza genome packaging. The Influenza virus genome is divided into eight RNA segments, and a long-standing question has been what is the mechanism for packaging all eight segments. Our hypothesis is that each of the eight viral RNAs (vRNA) are specifically packaged, and the removal of one vRNA will produce virus particles containing seven segments instead of eight. Virus particles missing a vRNA will be analyzed with EMT to determine the number of segments and which segments are packaged in each virus particle. Relevance: Viruses cause many human diseases throughout the world and result in major financial losses to society. In the United States Influenza is responsible for an estimated total direct and indirect cost of $10 billion per year, and the costs of a future pandemic could cost hundreds of billions of dollars. Investigating virus assembly and genome packaging will help researchers develop novel anti-viral targets.